Fatty acid desaturases and mutant sequences thereof

ABSTRACT

Seeds, plants and oils are provided having high oleic acid; low linoleic acid; and low linoleic acid plus linolenic acid; and advantageous functional or nutritional properties. Plants are disclosed that contain a mutation in a delta-12 or delta-15 fatty acid desaturase gene. Preferred plants are rapeseed and sunflower plants. Plants carrying such mutant genes have altered fatty acid composition in seeds. In one embodiment, a plant contains a mutation in a region having the conserved motif His-Xaa-Xaa-Xaa-His, found in delta-12 and delta-15 fatty acid desaturases. A preferred motif has the sequence His-Glu-Cys-Gly-His. A preferred mutation in this motif has the amino acid sequence His-Lys-Cys-Gly-His. Nucleic acid fragments are disclosed that comprise a mutant delta-12 or delta-15 fatty acid desaturase gene sequence.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of PCT application Ser. No.PCT/US96/20090, filed Dec. 13, 1996, which is a continuation-in-part ofU.S. application Ser. No. 08/572,027, filed Dec. 14, 1995.

TECHNICAL FIELD

This invention relates to fatty acid desaturases and nucleic acidsencoding desaturase proteins. More particularly, the invention relatesto nucleic acids encoding delta-12 and delta-15 fatty acid desaturaseproteins that affect fatty acid composition in plants, polypeptidesproduced from such nucleic acids and plants expressing such nucleicacids.

BACKGROUND OF THE INVENTION

Many breeding studies have been conducted to improve the fatty acidprofile of Brassica varieties. Pleines and Freidt, Fat Sci. Technol.,90(5), 167-171 (1988) describe plant lines with reduced C_(18:3) levels(2.5-5.8%) combined with high oleic content (73-79%). Rakow andMcGregor, J. Amer. Oil Chem. Soc., 50, 400-403 (Oct. 1973) discussproblems associated with selecting mutants for linoleic and linolenicacids. In. Can. J. Plant Sci., 68, 509-511 (Apr. 1988) Stellar summerrape producing seed oil with 3% linolenic acid and 28% linoleic acid isdisclosed. Roy and Tarr, Z. Pflanzenzuchtg, 95(3), 201-209 (1985)teaches transfer of genes through an interspecific cross from Brassicajuncea into Brassica napus resulting in a reconstituted line combininghigh linoleic with low linolenic acid content. Roy and Tarr, PlantBreeding, 98, 89-96 (1987) discuss prospects for development of B. napusL. having improved linolenic and linolenic acid content. European Patentapplication 323,753 published Jul. 12, 1989 discloses seeds and oilshaving greater than 79% oleic acid combined with less than 3.5%linolenic acid. Canvin, Can. J. Botany, 43, 63-69 (1965) discusses theeffect of temperature on the fatty acid composition of oils from severalseed crops including rapeseed.

Mutations typically are induced with extremely high doses of radiationand/or chemical mutagens (Gaul, H. Radiation Botany (1964) 4:155-232).High dose levels which exceed LD50, and typically reach LD90, led tomaximum achievable mutation rates. In mutation breeding of Brassicavarieties high levels of chemical mutagens alone or combined withradiation have induced a limited number of fatty acid mutations (Rakow,G. Z. Pflanzenzuchtg (1973) 69:62-82). The low α-linolenic acid mutationderived from the Rakow mutation breeding program did not have directcommercial application because of low seed yield. The first commercialcultivar using the low α-linolenic acid mutation derived in 1973 wasreleased in 1988 as the variety Stellar (Scarth, R. et al., Can. J.Plant Sci. (1988) 68:509-511). Stellar was 20% lower yielding thancommercial cultivars at the time of its release.

Alterations in fatty acid composition of vegetable oils is desirable formeeting specific food and industrial uses. For example, Brassicavarieties with increased monounsaturate levels (oleic acid) in the seedoil, and products derived from such oil, would improve lipid nutrition.Canola lines which are low in polyunsaturated fatty acids and high inoleic acid tend to have higher oxidative stability, which is a usefultrait for the retail food industry.

Delta-12 fatty acid desaturase (also known as oleic desaturase) isinvolved in the enzymatic conversion of oleic acid to linoleic acid.Delta-15 fatty acid desaturase (also known as linoleic acid desaturase)is involved in the enzymatic conversion of linoleic acid to α-linolenicacid. A microsomal delta-12 desaturase has been cloned and characterizedusing T-DNA tagging. Okuley, et al., Plant Cell 6:147-158 (1994). Thenucleotide sequences of higher plant genes encoding microsomal delta-12fatty acid desaturase are described in Lightner et al., WO94/11516.Sequences of higher plant genes encoding microsomal and plastid delta-15fatty acid desaturases are disclosed in Yadav, N., et al., PlantPhysiol., 103:467-476 (1993), WO 93/11245 and Arondel, V. et al.,Science, 258:1353-1355 (1992). However, there are no teachings thatdisclose mutations in delta-12 or delta-15 fatty acid desaturase codingsequences from plants. There is a need in the art for more efficientmethods to develop plant lines that contain delta-12 or delta-15 fattyacid desaturase gene sequence mutations effective for altering the fattyacid composition of seeds.

SUMMARY OF THE INVENTION

The invention comprises Brassicaceae or Helianthus seeds, plants andplant lines having at least one mutation that controls the levels ofunsaturated fatty acids in plants. One embodiment of the invention is anisolated nucleic acid fragment comprising a nucleotide sequence encodinga mutation from a mutant delta-12 fatty acid desaturase conferringaltered fatty composition in seeds when the fragment is present in aplant. A preferred sequence comprises a mutant sequence as shown in FIG.2. Another embodiment of the invention is an isolated nucleic acidfragment comprising a nucleotide sequence encoding a mutation from amutant delta-15 fatty acid desaturase. A plant in this embodiment may besoybean, oilseed Brassica species, sunflower, castor bean or corn. Themutant sequence may be derived from, for example, a Brassica napus,Brassica rapa, Brassica juncea or Helianthus delta-12 or delta-15desaturase gene.

Another embodiment of the invention involves a method of producing aBrassicaceae or Helianthus plant line comprising the steps of: (a)inducing mutagenesis in cells of a starting variety of a Brassicaceae orHelianthus species; (b) obtaining progeny plants from the mutagenizedcells; (c) identifying progeny plants that contain a mutation in adelta-12 or delta-15 fatty acid desaturase gene; and (d) producing aplant line by selfing or crossing. The resulting plant line may besubjected to mutagenesis in order to obtain a line having both adelta-12 desaturase mutation and a delta-15 desaturase mutation.

Yet another embodiment of the invention involves a method of producingplant lines containing altered fatty acid composition comprising: (a)crossing a first plant with a second plant having a mutant delta-12 ordelta-15 fatty acid desaturase; (b) obtaining seeds from the cross ofstep (a); (c) growing fertile plants from such seeds; (d) obtainingprogeny seed from the plants of step (c); and (e) identifying thoseseeds among the progeny that have altered fatty acid composition.Suitable plants are soybean, rapeseed, sunflower, safflower, castor beanand corn. Preferred plants are rapeseed and sunflower.

The invention is also embodied in vegetable oil obtained from plantsdisclosed herein, which vegetable oil has an altered fatty acidcomposition.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 shows a hypothetical DNA sequence of a Brassica Fad2 gene.SEQ ID NO:2 is the deduced amino acid sequence of SEQ ID NO:1.

SEQ ID NO:3 shows a hypothetical DNA sequence of a Brassica Fad2 genehaving a mutation at nucleotide 316. SEQ ID NO:4 is the deduced aminoacid sequence of SEQ ID NO:3.

SEQ ID NO:5 shows a hypothetical DNA sequence of a Brassica Fad2 gene.SEQ ID NO:6 is the deduced amino acid sequence of SEQ ID NO:5.

SEQ ID NO:7 shows a hypothetical DNA sequence of a Brassica Fad2 genehaving a mutation at nucleotide 515. SEQ ID NO:8 is the deduced aminoacid sequence of SEQ ID NO:7.

SEQ ID NO:9 shows the DNA sequence for the coding region of a wild typeBrassica Fad2-D gene. SEQ ID NO:10 is the deduced amino acid sequencefor SEQ ID NO:9.

SEQ ID NO:11 shows the DNA sequence for the coding region of the IMC 129mutant Brassica Fad2-D gene. SEQ ID NO:12 is the deduced amino acidsequence for SEQ ID NO:11.

SEQ ID NO:13 shows the DNA sequence for the coding region of a wild typeBrassica Fad2-F gene. SEQ ID NO:14 is the deduced amino acid sequencefor SEQ ID NO:13.

SEQ ID NO:15 shows the DNA sequence for the coding region of the Q508mutant Brassica Fad2-F gene. SEQ ID NO:16 is the deduced amino acidsequence for SEQ ID NO:15.

SEQ ID NO:17 shows the DNA sequence for the coding region of the Q4275mutant Brassica Fad2-F gene. SEQ ID NO:18 is the deduced amino acidsequence for SEQ ID NO:17.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a histogram showing the frequency distribution of seed oiloleic acid (C_(18:1)) content in a segregating population of a Q508 XWestar cross. The bar labeled WSGA 1A represents the C_(18:1) content ofthe Westar parent. The bar labeled Q508 represents the C_(18:1) contentof the Q508 parent.

FIG. 2 shows the nucleotide sequences for a Brassica Fad2-D wild typegene (Fad2-D wt), IMC129 mutant gene (Fad2-D GA316 IMC129), Fad2-F wildtype gene (Fad2-F wt), Q508 mutant gene (Fad2-F TA515 Q508) and Q4275mutant gene (Fad2-F GA908 Q4275).

FIG. 3 shows the deduced amino acid sequences for the polynucleotides ofFIG. 2.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

All percent fatty acids herein are percent by weight of the oil of whichthe fatty acid is a component.

As used herein, a “line” is a group of plants that display little or nogenetic variation between individuals for at least one trait. Such linesmay be created by several generations of self-pollination and selection,or vegetative propagation from a single parent using tissue or cellculture techniques. As used herein, the term “variety” refers to a linewhich is used for commercial production.

The term “mutagenesis” refers to the use of a mutagenic agent to inducerandom genetic mutations within a population of individuals. The treatedpopulation, or a subsequent generation of that population, is thenscreened for usable trait(s) that result from the mutations. A“population” is any group of individuals that share a common gene pool.As used herein “M₀” is untreated seed. As used herein, “M₁” is the seed(and resulting plants) exposed to a mutagenic agent, while “M₂” is theprogeny (seeds and plants) of self-pollinated M₁ plants, “M₃” is theprogeny of self-pollinated M₂ plants, and “M₄” is the progeny ofself-pollinated M₃ plants. “M₅” is the progeny of self-pollinated M₄plants. “M₆”, “M₇”, etc. are each the progeny of self-pollinated plantsof the previous generation. The term “selfed” as used herein meansself-pollinated.

“Stability” or “stable” as used herein means that with respect to agiven fatty acid component, the component is maintained from generationto generation for at least two generations and preferably at least threegenerations at substantially the same level, e.g., preferably ±5%. Themethod of invention is capable of creating lines with improved fattyacid compositions stable up to ±5% from generation to generation. Theabove stability may be affected by temperature, location, stress andtime of planting. Thus, comparison of fatty acid profiles should be madefrom seeds produced under similar growing conditions. Stability may bemeasured based on knowledge of prior generation.

Intensive breeding has produced Brassica plants whose seed oil containsless than 2% erucic acid. The same varieties have also been bred so thatthe defatted meal contains less than 30 μmol glucosinolates/gram.“Canola” as used herein refers to plant variety seed or oil whichcontains less than 2% erucic acid (C_(22:1)), and meal with less than 30μmol glucosinolates/gram.

Applicants have discovered plants with mutations in a delta-12 fattyacid desaturase gene. Such plants have useful alterations in the fattyacid compositions of the seed oil. Such mutations confer, for example,an elevated oleic acid content, a decreased, stabilized linoleic acidcontent, or both elevated oleic acid and decreased, stabilized linoleicacid content.

Applicants have further discovered plants with mutations in a delta-15fatty acid desaturase gene. Such plants have useful alterations in thefatty acid composition of the seed oil, e.g., a decreased, stabilizedlevel of α-linolenic acid.

Applicants have further discovered isolated nucleic acid fragments(polynucleotides) comprising sequences that carry mutations within thecoding sequence of delta-12 or delta-15 fatty acid desaturases. Themutations confer desirable alterations in fatty acid levels in the seedoil of plants carrying such mutations. Delta-12 fatty acid desaturase isalso known as omega-6 fatty acid desaturase and is sometimes referred toherein as Fad2 or 12-DES. Delta-15 fatty acid desaturase is also knownon omega-3 fatty acid desaturase and is sometimes referred to herein asFad3 or 15-DES.

A nucleic acid fragment of the invention may be in the form of RNA or inthe form of DNA, including cDNA, synthetic DNA or genomic DNA. The DNAmay be double-stranded or single-stranded, and if single-stranded, canbe either the coding strand or non-coding strand. An RNA analog may be,for example, mRNA or a combination of ribo- and deoxyribonucleotides.Illustrative examples of a nucleic acid fragment of the invention arethe mutant sequences shown in FIG. 3.

A nucleic acid fragment of the invention contains a mutation in amicrosomal delta-12 fatty acid desaturase coding sequence or a mutationin a microsomal delta-15 fatty acid desaturase coding sequence. Such amutation renders the resulting desaturase gene product non-functional inplants, relative to the function of the gene product encoded by thewild-type sequence. The non-functionality of the delta-12 desaturasegene product can be inferred from the decreased level of reactionproduct (linoleic acid) and increased level of substrate (oleic acid) inplant tissues expressing the mutant sequence, compared to thecorresponding levels in plant tissues expressing the wild-type sequence.The non-functionality of the delta-15 desaturase gene product can beinferred from the decreased level of reaction product (α-linolenic acid)and the increased level of substrate (linoleic acid) in plant tissuesexpressing the mutant sequence, compared to the corresponding levels inplant tissues expressing the wild-type sequence.

A nucleic acid fragment of the invention may comprise a portion of thecoding sequence, e.g., at least about 10 nucleotides, provided that thefragment contains at least one mutation in the coding sequence. Thelength of a desired fragment depends upon the purpose for which thefragment will be used, e.g., PCR primer, site-directed mutagenesis andthe like. In one embodiment, a nucleic acid fragment of the inventioncomprises the full length coding sequence of a mutant delta-12 or mutantdelta-15 fatty acid desaturase, e.g., the mutant sequences of FIG. 3. Inother embodiments, a nucleic acid fragment is about 20 to about 50nucleotides (or base pairs, bp), or about 50 to about 500 nucleotides,or about 500 to about 1200 nucleotides in length.

A mutation in a nucleic acid fragment of the invention may be in anyportion of the coding sequence that renders the resulting gene productnon-functional. Suitable types of mutations include, without limitation,insertions of nucleotides, deletions of nucleotides, or transitions andtransversions in the wild-type coding sequence. Such mutations result ininsertions of one or more amino acids, deletions of one or more aminoacids, and non-conservative amino acid substitutions in thecorresponding gene product. In some embodiments, the sequence of anucleic acid fragment may comprise more than one mutation or more thanone type of mutation.

Insertion or deletion of amino acids in a coding sequence may, forexample, disrupt the conformation of essential alpha-helical orbeta-pleated sheet regions of the resulting gene product. Amino acidinsertions or deletions may also disrupt binding or catalytic sitesimportant for gene product activity. It is known in the art that theinsertion or deletion of a larger number of contiguous amino acids ismore likely to render the gene product non-functional, compared to asmaller number of inserted or deleted amino acids.

Non-conservative amino acid substitutions may replace an amino acid ofone class with an amino acid of a different class. Non-conservativesubstitutions may make a substantial change in the charge orhydrophobicity of the gene product. Non-conservative amino acidsubstitutions may also make a substantial change in the bulk of theresidue side chain, e.g., substituting an alanyl residue for a isoleucylresidue.

Examples of non-conservative substitutions include the substitution of abasic amino acid for a non-polar amino acid, or a polar amino acid foran acidic amino acid. Because there are only 20 amino acids encoded in agene, substitutions that result in a non-functional gene product may bedetermined by routine experimentation, incorporating amino acids of adifferent class in the region of the gene product targeted for mutation.

Preferred mutations are in a region of the nucleic acid encoding anamino acid sequence motif that is conserved among delta-12 fatty aciddesaturases or delta-15 fatty acid desaturases, such as aHis-Xaa-Xaa-Xaa-His motif (Tables 1-3). An example of a suitable regionhas a conserved HECGH motif that is found, for example, in nucleotidescorresponding to amino acids 105 to 109 of the Arabidopsis and Brassicadelta-12 desaturase sequences, in nucleotides corresponding to aminoacids 101 to 105 of the soybean delta-12 desaturase sequence and innucleotides corresponding to amino acids 111 to 115 of the maizedelta-12 desaturase sequence. See e.g., WO 94/115116; Okuley et al.,Plant Cell 6:147-158 (1994). The one letter amino acid designations usedherein are described in Alberts, B. et al., Molecular Biology of theCell, 3rd edition, Garland Publishing, New York, 1994. Amino acidsflanking this motif are also highly conserved among delta-12 anddelta-15 desaturases and are also suitable candidates for mutations infragments of the invention.

An illustrative embodiment of a mutation in a nucleic acid fragment ofthe invention is a Glu to Lys substitution in the HECGH motif of aBrassica microsomal delta-12 desaturase sequence, either the D form orthe F form. This mutation results in the sequence HECGH being changed toHKCGH as seen by comparing SEQ ID NO:10 (wild-type D form) to SEQ IDNO:12 (mutant D form). A similar mutation in other Fad-2 sequences iscontemplated to result in a non-functional gene product. (Compare SEQ IDNO:2 to SEQ ID NO:4).

A similar motif may be found at amino acids 101 to 105 of theArabidopsis microsomal delta-15 fatty acid desaturase, as well as in thecorresponding rape and soybean desaturases (Table 5). See, e.g., WO93/11245; Arondel, V. et al., Science, 258:1153-1155 (1992); Yadav, N.et al., Plant Physiol., 103:467-476 (1993). Plastid delta-15 fatty acidshave a similar motif (Table 5).

Among the types of mutations in an HECGH motif that render the resultinggene product non-functional are non-conservative substitutions. Anillustrative example of a non-conservative substitution is substitutionof a glycine residue for either the first or second histidine. Such asubstitution replaces a charged residue (histidine) with a non-polarresidue (glycine). Another type of mutation that renders the resultinggene product non-functional is an insertion mutation, e.g., insertion ofa glycine between the cysteine and glutamic acid residues in the HECGHmotif.

Other regions having suitable conserved amino acid motifs include theHRRHH motif shown in Table 2, the HRTHH motif shown in Table 6 and theHVAHH motif shown in Table 3. See, e.g., WO 94/115116; Hitz, W. et al.,Plant Physiol., 105:635-641 (1994); Okuley, J., et al., supra; andYadav, N. et al., supra. An illustrative example of a mutation in theregion shown in Table 3 is a mutation at nucleotides corresponding tothe codon for glycine (amino acid 303 of B. napus). A non-conservativeGly to Glu substitution results in the amino acid sequence DRDYGILNKVbeing changed to sequence DRDYEILNKV (compare wild-type F form SEQ IDNO:14 to mutant Q4275 SEQ ID NO:18, FIG. 3).

Another region suitable for a mutation in a delta-12 desaturase sequencecontains the motif KYLNNP at nucleotides corresponding to amino acids171 to 175 of the Brassica desaturase sequence. An illustrative exampleof a mutation is this region is a Leu to His substitution, resulting inthe amino acid sequence (Table 4) KYHNN (compare wild-type Fad2-F SEQ IDNO:14 to mutant SEQ ID NO:16). A similar mutation in other Fad-2 aminoacid sequences is contemplated to result in a non-functional geneproduct. (Compare SEQ ID NO:6 to SEQ ID NO:8). TABLE 1 Alignment ofAmino Acid Sequences from Microsomal Delta-12 Fatty Acid DesaturasesSpecies Position Amino Acid Sequence Arabidopsis thaliana 100-129IWVIAHECGH HAFSDYQWLD DTVGLIFHSF Glycine max  96-125 VWVIAHECGHHAFSKYQWVD DVVGLTLHST Zea mays 106-135 VWVIAHECGH HAFSDYSLLD DVVGLVLHSSRicinus communis ^(a)  1-29 WVMAHDCGH HAFSDYQLLD DVVGLILHSC Brassicanapus D 100-128 VWVIAHECGH HAFSDYQWLD DTVGLIFHS Brassica napus F 100-128VWVIAHECGH HAFSDYQWLD DTVGLIFHS^(a)from plasmid pRF2-1C

TABLE 2 Alignment of Amino Acid Sequences from Microsomal Delta-12 FattyAcid Desaturases Species Position Amino Acid Sequence Arabidopsisthaliana 130-158 LLVPYFSWKY SHRRHHSNTG SLERDEVFV Glycine max 126-154LLVPYFSWKI SHRRHHSNTG SLDRDEVFV Zea mays 136-164 LMVPYFSWKY SHRRHHSNTGSLERDEVFV Ricinus communis ^(a) 30-58 LLVPYFSWKH SHRRHHSNTG SLERDEVFVBrassica napus D 130-158 LLVPYFSWKY SHRRHHSNTG SLERDEVFV Brassica napusF 130-158 LLVPYFSWKY SHRRHHSNTG SLERDEVFV^(a)from plasmid pRF2-1C

TABLE 3 Alignment of Amino Acid Sequences from Microsomal Delta-12 FattyAcid Desaturases Species Position Amino Acid Sequence Arabidopsisthaliana 298-333 DRDYGILNKV FHNITDTHVA HHLFSTMPHY NAMEAT Glycine max294-329 DRDYGILNKV FHHITDTHVA HHLFSTMPHY HAMEAT Zea mays 305-340DRDYGILNRV FHNITDTHVA HHLFSTMPHY HAMEAT Ricinus communis ^(a) 198-224DRDYGILNKV FHNITDTQVA HHLF TMP Brassica napus D 299-334 DRDYGILNKVFHNITDTHVA HHLFSTMPHY HAMEAT Brassica napus F 299-334 DRDYGILNKVFHNITDTHVA HHLFSTMPHY HAMEAT^(a)from plasmid pRF2-1C

TABLE 4 Alignment of Conserved Amino Acids from Microsomal Delta-12Fatty Acid Desaturases Species Position Amino Acid Sequence Arabidopsisthaliana 165-180 IKWYGKYLNN PLGRIM Glycine max 161-176 VAWFSLYLNN PLGRAVZea mays 172-187 PWYTPYVYNN PVGRVV Ricinus communis ^(a) 65-80IRWYSKYLNN PPGRIM Brassica napus D 165-180 IKWYGKYLNN PLGRTV Brassicanapus F 165-180 IKWYGKYLNN PLGRTV^(a)from plasmid pRF2-1C

TABLE 5 Alignment of Conserved Amino Acids from Plastid and MicrosomalDelta-15 Fatty Acid Desaturases Species Position Amino Acid SequenceArabidopsis thaliana ^(a) 156-177 WALFVLGHD CGHGSFSNDP KLN Brassicanapus ^(a) 114-135 WALFVLGHD CGHGSFSNDP RLN Glycine max ^(a) 164-185WALFVLGHD CGHGSFSNNS KLN Arabidopsis thaliana  94-115 WAIFVLGHDCGHGSFSDIP LLN Brassica napus  87-109 WALFVLGHD CGHGSFSNDP RLN Glycinemax  93-114 WALFVLGHD CGHGSFSDSP PLN^(a)Plastid sequences

TABLE 6 Alignment of Conserved Amino Acids from Plastid and MicrosomalDelta-15 Fatty Acid Desaturases Species Position Amino Acid Sequence A.thaliana ^(a) 188-216 ILVPYHGWRI SHRTHHQNHG HVENDESWH B. napus ^(a)146-174 ILVPYHGWRI SHRTHHQNHG HVENDESWH Glycine max ^(a) 196-224ILVPYHGWRI SHRTHHQHHG HAENDESWH A. thaliana 126-154 ILVPYHGWRISHRTHHQNHG HVENDESWV Brassica napus 117-145 ILVPYHGWRI SHRTHHQNHGHVENDESWV Glycine max 125-153 ILVPYHGWRI SHRTHHQNHG HIEKDESWV^(a)Plastid sequences

The conservation of amino acid motifs and their relative positionsindicates that regions of a delta-12 or delta-15 fatty acid desaturasethat can be mutated in one species to generate a non-functionaldesaturase can be mutated in the corresponding region from other speciesto generate a non-functional delta-12 desaturase or delta-15 desaturasegene product in that species.

Mutations in any of the regions of Tables 1-6 are specifically includedwithin the scope of the invention and are substantially identical tothose mutations exemplified herein, provided that such mutation (ormutations) renders the resulting desaturase gene product non-functional,as discussed hereinabove.

A nucleic acid fragment containing a mutant sequence can be generated bytechniques known to the skilled artisan. Such techniques include,without limitation, site-directed mutagenesis of wild-type sequences anddirect synthesis using automated DNA synthesizers.

A nucleic acid fragment containing a mutant sequence can also begenerated by mutagenesis of plant seeds or regenerable plant tissue by,e.g., ethyl methane sulfonate, X-rays or other mutagens. Withmutagenesis, mutant plants having the desired fatty acid phenotype inseeds are identified by known techniques and a nucleic acid fragmentcontaining the desired mutation is isolated from genomic DNA or RNA ofthe mutant line. The site of the specific mutation is then determined bysequencing the coding region of the delta-12 desaturase or delta-15desaturase gene. Alternatively, labeled nucleic acid probes that arespecific for desired mutational events can be used to rapidly screen amutagenized population.

The disclosed method may be applied to all oilseed Brassica species, andto both Spring and Winter maturing types within each species. Physicalmutagens, including but not limited to X-rays, UV rays, and otherphysical treatments which cause chromosome damage, and other chemicalmutagens, including but not limited to ethidium bromide,nitrosoguanidine, diepoxybutane etc. may also be used to inducemutations. The mutagenesis treatment may also be applied to other stagesof plant development, including but not limited to cell cultures,embryos, microspores and shoot apices.

“Stable mutations” as used herein are defined as M₅ or more advancedlines which maintain a selected altered fatty acid profile for a minimumof three generations, including a minimum of two generations under fieldconditions, and exceeding established statistical thresholds for aminimum of two generations, as determined by gas chromatographicanalysis of a minimum of 10 randomly selected seeds bulked together.Alternatively, stability may be measured in the same way by comparing tosubsequent generations. In subsequent generations, stability is definedas having similar fatty acid profiles in the seed as that of the prioror subsequent generation when grown under substantially similarconditions.

Mutation breeding has traditionally produced plants carrying, inaddition to the trait of interest, multiple, deleterious traits, e.g.,reduced plant vigor and reduced fertility. Such traits may indirectlyaffect fatty acid composition, producing an unstable mutation; and/orreduce yield, thereby reducing the commercial utility of the invention.To eliminate the occurrence of deleterious mutations and reduce the loadof mutations carried by the plant, a low mutagen dose is used in theseed treatments to create an LD30 population. This allows for the rapidselection of single gene mutations for fatty acid traits in agronomicbackgrounds which produce acceptable yields.

The seeds of several different fatty acid lines have been deposited withthe American Type Culture Collection and have the following accessionnumbers. Line Accession No. Deposit Date A129.5 40811 May 25, 1990A133.1 40812 May 25, 1990 M3032.1 75021 Jun. 7, 1991 M3062.8 75025 Jun.7, 1991 M3028.10 75026 Jun. 7, 1991 IMC130 75446 Apr. 16, 1993 Q427597569 May 10, 1996

In some plant species or varieties more than one form of endogenousmicrosomal delta-12 desaturase may be found. In amphidiploids, each formmay be derived from one of the parent genomes making up the speciesunder consideration. Plants with mutations in both forms have a fattyacid profile that differs from plants with a mutation in only one form.An example of such a plant is Brassica napus line Q508, adoubly-mutagenized line containing a mutant D-form of delta-12desaturase (SEQ ID NO:11) and a mutant F-form of delta-12 desaturase(SEQ ID NO:15). Another example is line Q4275, which contains a mutantD-form of delta-12 desaturase (SEQ ID NO:11) and a mutant F-form ofdelta-12 desaturase (SEQ ID NO:17). See FIGS. 2-3.

Preferred host or recipient organisms for introduction of a nucleic acidfragment of the invention are the oil-producing species, such as soybean(Glycine max), rapeseed (e.g., Brassica napus, B. rapa and B. juncea),sunflower (Helianthus annus), castor bean (Ricinus communis), corn (Zeamays), and safflower (Carthamus tinctorius).

A nucleic acid fragment of the invention may further comprise additionalnucleic acids. For example, a nucleic acid encoding a secretory orleader amino acid sequence can be linked to a mutant desaturase nucleicacid fragment such that the secretory or leader sequence is fusedin-frame to the amino terminal end of a mutant delta-12 or delta-15desaturase polypeptide. Other nucleic acid fragments are known in theart that encode amino acid sequences useful for fusing in-frame to themutant desaturase polypeptides disclosed herein. See, e.g., U.S. Pat.No. 5,629,193 incorporated herein by reference. A nucleic acid fragmentmay also have one or more regulatory elements operably linked thereto.

The present invention also comprises nucleic acid fragments thatselectively hybridize to mutant desaturase sequences. Such a nucleicacid fragment typically is at least 15 nucleotides in length.Hybridization typically involves Southern analysis (Southern blotting),a method by which the presence of DNA sequences in a target nucleic acidmixture are identified by hybridization to a labeled oligonucleotide orDNA fragment probe. Southern analysis typically involves electrophoreticseparation of DNA digests on agarose gels, denaturation of the DNA afterelectrophoretic separation, and transfer of the DNA to nitrocellulose,nylon, or another suitable membrane support for analysis with aradiolabeled, biotinylated, or enzyme-labeled probe as described insections 9.37-9.52 of Sambrook et al., (1989) Molecular Cloning, secondedition, Cold Spring Harbor Laboratory, Plainview; N.Y.

A nucleic acid fragment can hybridize under moderate stringencyconditions or, preferably, under high stringency conditions to a mutantdesaturase sequence. High stringency conditions are used to identifynucleic acids that have a high degree of homology to the probe. Highstringency conditions can include the use of low ionic strength and hightemperature for washing, for example, 0.015 M NaCl/0.0015 M sodiumcitrate (0.1×SSC); 0.1% sodium lauryl sulfate (SDS) at 50-65° C.Alternatively, a denaturing agent such as formamide can be employedduring hybridization, e.g., 50% formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.Another example is the use of 50% formamide, 5×SSC (0.75 M NaCl, 0.075 Msodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC and 0.1% SDS.

Moderate stringency conditions refers to hybridization conditions usedto identify nucleic acids that have a lower degree of identity to theprobe than do nucleic acids identified under high stringency conditions.Moderate stringency conditions can include the use of higher ionicstrength and/or lower temperatures for washing of the hybridizationmembrane, compared to the ionic strength and temperatures used for highstringency hybridization. For example, a wash solution comprising 0.060M NaCl/0.0060 M sodium citrate (4×SSC) and 0.1% sodium lauryl sulfate(SDS) can be used at 50° C., with a last wash in 1×SSC, at 65° C.Alternatively, a hybridization wash in 1×SSC at 37° C. can be used.

Hybridization can also be done by Northern analysis (Northern blotting),a method used to identify RNAs that hybridize to a known probe such asan oligonucleotide, DNA fragment, cDNA or fragment thereof, or RNAfragment. The probe is labeled with a radioisotope such as ³²P, bybiotinylation or with an enzyme. The RNA to be analyzed can be usuallyelectrophoretically separated on an agarose or polyacrylamide gel,transferred to nitrocellulose, nylon, or other suitable membrane, andhybridized with the probe, using standard techniques well known in theart such as those described in sections 7.39-7.52 of Sambrook et al.,supra.

A polypeptide of the invention comprises an isolated polypeptide havinga mutant amino acid sequence, as well as derivatives and analogsthereof. See, e.g., the mutant amino acid sequences of FIG. 3. By“isolated” is meant a polypeptide that is expressed and produced in anenvironment other than the environment in which the polypeptide isnaturally expressed and produced. For example, a plant polypeptide isisolated when expressed and produced in bacteria or fungi. A polypeptideof the invention also comprises variants of the mutant desaturasepolypeptides disclosed herein, as discussed above.

In one embodiment of the claimed invention, a plant contains both adelta-12 desaturase mutation and a delta-15 desaturase mutation. Suchplants can have a fatty acid composition comprising very high oleic acidand very low alpha-linolenic acid levels. Mutations in delta-12desaturase and delta-15 desaturase may be combined in a plant by makinga genetic cross between delta-12 desaturase and delta-15 desaturasesingle mutant lines. A plant having a mutation in delta-12 fatty aciddesaturase is crossed or mated with a second plant having a mutation indelta-15 fatty acid desaturase. Seeds produced from the cross areplanted and the resulting plants are selfed in order to obtain progenyseeds. These progeny seeds are then screened in order to identify thoseseeds carrying both mutant genes.

Alternatively, a line possessing either a delta-12 desaturase or adelta-15 desaturase mutation can be subjected to mutagenesis to generatea plant or plant line having mutations in both delta-12 desaturase anddelta-15 desaturase. For example, the IMC 129 line has a mutation in thecoding region (Glu₁₀₆ to Lys₁₀₆) of the D form of the microsomaldelta-12 desaturase structural gene. Cells (e.g., seeds) of this linecan be mutagenized to induce a mutation in a delta-15 desaturase gene,resulting in a plant or plant line carrying a mutation in a delta-12fatty acid desaturase gene and a mutation in a delta-15 fatty aciddesaturase gene.

Progeny includes descendants of a particular plant or plant line, e.g.,seeds developed on an instant plant are descendants. Progeny of aninstant plant include seeds formed on F₁, F₂, F₃, and subsequentgeneration plants, or seeds formed on BC₁, BC₂, BC₃ and subsequentgeneration plants.

Plants according to the invention preferably contain an altered fattyacid composition. For example, oil obtained from seeds of such plantsmay have from about 69 to about 90% oleic acid, based on the total fattyacid composition of the seed. Such oil preferably has from about 74 toabout 90% oleic acid, more preferably from about 80 to about 90% oleicacid. In some embodiments, oil obtained from seeds produced by plants ofthe invention may have from about 2.0% to about 5.0% saturated fattyacids, based on total fatty acid composition of the seeds. In someembodiments, oil obtained from seeds of the invention may have fromabout 1.0% to about 14.0% linoleic acid, or from about 0.5% to about10.0% α-linolenic acid.

Oil composition typically is analyzed by crushing and extracting fattyacids from bulk seed samples (e.g., 10 seeds). Fatty acid triglyceridesin the seed are hydrolyzed and converted to fatty acid methyl esters.Those seeds having an altered fatty acid composition may be identifiedby techniques known to the skilled artisan, e.g., gas-liquidchromatography (GLC) analysis of a bulked seed sample or of a singlehalf-seed. Half-seed analysis is well known in the art to be usefulbecause the viability of the embryo is maintained and thus those seedshaving a desired fatty acid profile may be planted to from the nextgeneration. However, half-seed analysis is also known to be aninaccurate representation of genotype of the seed being analyzed. Bulkseed analysis typically yields a more accurate representation of thefatty acid profile of a given genotype. Fatty acid composition can alsobe determined on larger samples, e.g., oil obtained by pilot plant orcommercial scale refining, bleaching and deodorizing of endogenous oilin the seeds.

The nucleic acid fragments of the invention can be used as markers inplant genetic mapping and plant breeding programs. Such markers mayinclude restriction fragment length polymorphism (RFLP), randomamplification polymorphism detection (RAPD), polymerase chain reaction(PCR) or self-sustained sequence replication (3SR) markers, for example.Marker-assisted breeding techniques may be used to identify and follow adesired fatty acid composition during the breeding process.Marker-assisted breeding techniques may be used in addition to, or as analternative to, other sorts of identification techniques. An example ofmarker-assisted breeding is the use of PCR primers that specificallyamplify a sequence containing a desired mutation in delta-12 desaturaseor delta-15 desaturase.

Methods according to the invention are useful in that the resultingplants and plant lines have desirable seed fatty acid compositions aswell as superior agronomic properties compared to known lines havingaltered seed fatty acid composition. Superior agronomic characteristicsinclude, for example, increased seed germination percentage, increasedseedling vigor, increased resistance to seedling fungal diseases(damping off, root rot and the like), increased yield, and improvedstandability.

While the invention is susceptible to various modifications andalternative forms, certain specific embodiments thereof are described inthe general methods and examples set forth below. For example theinvention may be applied to all Brassica species, including B. rapa, B.juncea, and B. hirta, to produce substantially similar results. Itshould be understood, however, that these examples are not intended tolimit the invention to the particular forms disclosed but, instead theinvention is to cover all modifications, equivalents and alternativesfalling within the scope of the invention. This includes the use ofsomaclonal variation; physical or chemical mutagenesis of plant parts;anther, microspore or ovary culture followed by chromosome doubling; orself- or cross-pollination to transmit the fatty acid trait, alone or incombination with other traits, to develop new Brassica lines.

EXAMPLE 1 Mutagenesis

Seeds of Westar, a Canadian (Brassica napus) spring canola variety, weresubjected to chemical mutagenesis. Westar is a registered Canadianspring variety with canola quality. The fatty acid composition offield-grown Westar, 3.9% C_(16:0), 1.9% C_(18:1), 67.5% C_(18:1), 17.6%C_(18:2), 7.4% C_(18:3), <2% C20:1+C_(22:1), has remained stable undercommercial production, with <±10% deviation, since 1982.

Prior to mutagenesis, 30,000 seeds of B. napus cv. Westar seeds werepreimbibed in 300-seed lots for two hours on wet filter paper to softenthe seed coat. The preimbibed seeds were placed in 80 mMethylmethanesulfonate (EMS) for four hours. Following mutagenesis, theseeds were rinsed three times in distilled water. The seeds were sown in48-well flats containing Pro-Mix. Sixty-eight percent of the mutagenizedseed germinated. The plants were maintained at 25° C./15° C., 14/10 hrday/night conditions in the greenhouse. At flowering, each plant wasindividually self-pollinated.

M₂ seed from individual plants were individually catalogued and stored,approximately 15,000 M₂ lines was planted in a summer nursery in Carman,Manitoba. The seed from each selfed plant were planted in 3-meter rowswith 6-inch row spacing. Westar was planted as the check variety.Selected lines in the field were selfed by bagging the main raceme ofeach plant. At maturity, the selfed plants were individually harvestedand seeds were catalogued and stored to ensure that the source of theseed was known.

Self-pollinated M₃ seed and Westar controls were analyzed in 10-seedbulk samples for fatty acid composition via gas chromatography.Statistical thresholds for each fatty acid component were establishedusing a Z-distribution with a stringency level of 1 in 10,000. Mean andstandard deviation values were determined from the non-mutagenizedWestar control population in the field. The upper and lower statisticalthresholds for each fatty acid were determined from the mean value ofthe population ± the standard deviation, multiplied by theZ-distribution. Based on a population size of 10,000, the confidenceinterval is 99.99%.

The selected M₃ seeds were planted in the greenhouse along with Westarcontrols. The seed was sown in 4-inch pots containing Pro-Mix soil andthe plants were maintained at 25° C./15° C., 14/10 hr day/night cycle inthe greenhouse. At flowering, the terminal raceme was self-pollinated bybagging. At maturity, selfed M₄ seed was individually harvested fromeach plant, labelled, and stored to ensure that the source of the seedwas known.

The M₄ seed was analyzed in 10-seed bulk samples. Statistical thresholdsfor each fatty acid component were established from 259 control samplesusing a Z-distribution of 1 in 800. Selected M₄ lines were planted in afield trial in Carman, Manitoba in 3-meter rows with 6-inch spacing. TenM₄ plants in each row were bagged for self-pollination. At maturity, theselfed plants were individually harvested and the open pollinated plantsin the row were bulk harvested. The M₅ seed from single plant selectionswas analyzed in 10-seed bulk samples and the bulk row harvest in 50-seedbulk samples.

Selected M₅ lines were planted in the greenhouse along with Westarcontrols. The seed was grown as previously described. At flowering theterminal raceme was self-pollinated by bagging. At maturity, selfed M₆seed was individually harvested from each plant and analyzed in 10-seedbulk samples for fatty acid composition.

Selected M₆ lines were entered into field trials in Eastern Idaho. Thefour trial locations were selected for the wide variability in growingconditions. The locations included Burley, Tetonia, Lamont and Shelley(Table 7). The lines were planted in four 3-meter rows with an 8-inchspacing, each plot was replicated four times. The planting design wasdetermined using a Randomized Complete Block Designed. The commercialcultivar Westar was used as a check cultivar. At maturity the plots wereharvested to determine yield. Yield of the entries in the trial wasdetermined by taking the statistical average of the four replications.The Least Significant Difference Test was used to rank the entries inthe randomized complete block design. TABLE 7 Trial Locations forSelected Fatty Acid Mutants LOCATION SITE CHARACTERIZATIONS BURLEYIrrigated. Long season. High temperatures during flowering. TETONIADryland. Short season. Cool temperatures. LAMONT Dryland. Short season.Cool temperatures. SHELLEY Irrigated. Medium season. High temperaturesduring flowering.

To determine the fatty acid profile of entries, plants in each plot werebagged for self-pollination. The M₇ seed from single plants was analyzedfor fatty acids in ten-seed bulk samples.

To determine the genetic relationships of the selected fatty acidmutants crosses were made. Flowers of M₆ or later generation mutationswere used in crossing. F₁ seed was harvested and analyzed for fatty acidcomposition to determine the mode of gene action. The F₁ progeny wereplanted in the greenhouse. The resulting plants were self-pollinated,the F₂ seed harvested and analyzed for fatty acid composition forallelism studies. The F₂ seed and parent line seed was planted in thegreenhouse, individual plants were self-pollinated. The F₃ seed ofindividual plants was tested for fatty acid composition using 10-seedbulk samples as described previously.

In the analysis of some genetic relationships dihaploid populations weremade from the microspores of the F₁ hybrids. Self-pollinated seed fromdihaploid plants were analyzed for fatty acid analysis using methodsdescribed previously.

For chemical analysis, 10-seed bulk samples were hand ground with aglass rod in a 15-mL polypropylene tube and extracted in 1.2 mL 0.25 NKOH in 1:1 ether/methanol. The sample was vortexed for 30 sec. andheated for 60 sec. in a 60° C. water bath. Four mL of saturated NaCl and2.4 mL of iso-octane were added, and the mixture was vortexed again.After phase separation, 600 μL of the upper organic phase were pipettedinto individual vials and stored under nitrogen at −5° C. One μL sampleswere injected into a Supelco SP-2330 fused silica capillary column (0.25mm ID, 30 M length, 0.20 μm df).

The gas chromatograph was set at 180° C. for 5.5 minutes, thenprogrammed for a 2° C./minute increase to 212° C., and held at thistemperature for 1.5 minutes. Total run time was 23 minutes.Chromatography settings were: Column head pressure—15 psi, Column flow(He)—0.7 mL/min., Auxiliary and Column flow—33 mL/min., Hydrogen flow—33mL/min., Air flow—400 mL/min., Injector temperature—250° C., Detectortemperature—300° C., Split vent—1/15.

Table 8 describes the upper and lower statistical thresholds for eachfatty acid of interest. TABLE 8 Statistical Thresholds for SpecificFatty Acids Derived from Control Westar Plantings Percent Fatty AcidsGenotype C_(16:0) C_(18:0) C_(18:1) C_(18:2) C_(18:3) Sats* M₃Generation (1 in 10,000 rejection rate) Lower 3.3 1.4 — 13.2 5.3 6.0Upper 4.3 2.5 71.0 21.6 9.9 8.3 M₄ Generation (1 in 800 rejection rate)Lower 3.6 0.8 — 12.2 3.2 5.3 Upper 6.3 3.1 76.0 32.4 9.9 11.2 M₅Generation (1 in 755 rejection rate) Lower 2.7 0.9 — 9.6 2.6 4.5 Upper5.7 2.7 80.3 26.7 9.6 10.0*Sats = Total Saturate Content

EXAMPLE 2 High Oleic Acid Canola Lines

In the studies of Example 1, at the M₃ generation, 31 lines exceeded theupper statistical threshold for oleic acid (≧71.0%). Line W7608.3 had71.2% oleic acid. At the M₄ generation, its selfed progeny (W7608.3.5,since designated A129.5) continued to exceed the upper statisticalthreshold for C_(18:1) with 78.8% oleic acid. M₅ seed of fiveself-pollinated plants of line A129.5 (ATCC 40811) averaged 75.0% oleicacid. A single plant selection, A129.5.3 had 75.6% oleic acid. The fattyacid composition of this high oleic acid mutant, which was stable underboth field and greenhouse conditions to the M, generation, is summarizedin Table 9. This line also stably maintained its mutant fatty acidcomposition to the M₇ generation in field trials in multiple locations.Over all locations the self-pollinated plants (A129) averaged 78.3%oleic acid. The fatty acid composition of the A129 for each Idaho triallocation are summarized in Table 10. In multiple location replicatedyield trials, A129 was not significantly different in yield from theparent cultivar Westar.

The canola oil of A129, after commercial processing, was found to havesuperior oxidative stability compared to Westar when measured by theAccelerated Oxygen Method (AOM), American Oil Chemists' Society OfficialMethod Cd 12-57 for fat stability; Active Oxygen Method (revised 1989).The AOM of Westar was 18 AOM hours and for A129 was 30 AOM hours. TABLE9 Fatty Acid Composition of a High Oleic Acid Canola Line Produced bySeed Mutagenesis Percent Fatty Acids Genotype C_(16:0) C_(18:0) C_(18:1)C_(18:2) C_(18:3) Sats Westar 3.9 1.9 67.5 17.6 7.4 7.0 W7608.3 3.9 2.471.2 12.7 6.1 7.6 (M₃) W7608.3.5 3.9 2.0 78.8 7.7 3.9 7.3 (M₄) A129.5.33.8 2.3 75.6 9.5 4.9 7.6 (M₅)Sats = Total Saturate Content

TABLE 10 Fatty Acid Composition of a Mutant High Oleic Acid Line atDifferent Field Locations in Idaho Percent Fatty Acids Location C_(16:0)C_(18:0) C_(18:1) C_(18:2) C_(18:3) Sats Burley 3.3 2.1 77.5 8.1 6.0 6.5Tetonia 3.5 3.4 77.8 6.5 4.7 8.5 Lamont 3.4 1.9 77.8 7.4 6.5 6.3 Shelley3.3 2.6 80.0 5.7 4.5 7.7Sats = Total Saturate Content

The genetic relationship of the high oleic acid mutation A129 to otheroleic desaturases was demonstrated in crosses made to commercial canolacultivars and a low linolenic acid mutation. A129 was crossed to thecommercial cultivar Global (C_(16:0)—4.5%, C_(18:0)—1.5%,C_(18:1)—62.9%, C_(18:2)—20.0%, C_(18:3)—7.3%). Approximately 200 F₂individuals were analyzed for fatty acid composition. The results aresummarized in Table 11. The segregation fit 1:2:1 ratio suggesting asingle co-dominant gene controlled the inheritance of the high oleicacid phenotype. TABLE 11 Genetic Studies of A129 X Global FrequencyC_(18:0) Genotype Content (%) Observed Expected od − od− 77.3  43 47 od− od+ 71.7 106 94 od + od+ 66.1  49 47

A cross between A129 and IMC 01, a low linolenic acid variety(C_(16:0)—4.1%, C_(18:0)—1.9%, C_(18:1)—66.4%, C_(18:2)—18.1%,C_(18:3)—5.7%), was made to determine the inheritance of the oleic aciddesaturase and linoleic acid desaturase. In the F₁ hybrids both theoleic acid and linoleic acid desaturase genes approached the mid-parentvalues indicating a co-dominant gene actions. Fatty acid analysis of theF₂ individuals confirmed a 1:2:1:2:4:2:1:2:1 segregation of twoindependent, co-dominant genes (Table 12). A line was selected from thecross of A129 and IMC01 and designated as IMC130 (ATCC deposit no.75446) as described in U.S. patent application Ser. No. 08/425,108,incorporated herein by reference. TABLE 12 Genetic Studies of A129 X IMC01 Frequency Genotype Ratio Observed Expected od − od − ld − ld− 1 11 12od − od − ld − ld+ 2 30 24 od − od − ld + ld+ 1 10 12 od − od + ld − ld−2 25 24 od − od + ld − ld+ 4 54 47 od − od + ld + ld+ 2 18 24 od + od +ld − ld− 1  7 12 od + od + ld − ld+ 2 25 24 od + od + ld + ld+ 1  8 12

An additional high oleic acid line, designated A128.3, was also producedby the disclosed method. A 50-seed bulk analysis of this line showed thefollowing fatty acid composition: C_(16:0)—3.5%, C_(18:0)—1.8%,C_(18:1)—77.3%, C_(18:2)—9.0%, C_(18:3)—5.6%, FDA Sats—5.3%, TotalSats—6.4%. This line also stably maintained its mutant fatty acidcomposition to the M₇ generation. In multiple locations replicated yieldtrials, A128 was not significantly different in yield from the parentcultivar Westar.

A129 was crossed to A128.3 for allelism studies. Fatty acid compositionof the F₂ seed showed the two lines to be allelic. The mutational eventsin A129 and A128.3 although different in origin were in the same gene.

An additional high oleic acid line, designated M3028.-10 (ATCC 75026),was also produced by the disclosed method in Example 1. A 10-seed bulkanalysis of this line showed the following fatty acid composition:C_(16:0)—3.5%, C_(18:0)—1.8%, C_(18:1)—77.3%, C_(18:2)—9.0%,C_(18:3)—5.6%, FDA Saturates—5.3%, Total Saturates—6.4%. In a singlelocation replicated yield trial M3028.10 was not significantly differentin yield from the parent cultivar Westar.

EXAMPLE 3 Low Linoleic Acid Canola

In the studies of Example 1, at the M₃ generation, 80 lines exceeded thelower statistical threshold for linoleic acid (≦13.2%). Line W12638.8had 9.4% linoleic acid. At the M₄ and M₅ generations, its selfedprogenies [W12638.8, since designated A133.1 (ATCC 40812)] continued toexceed the statistical threshold for low C_(18:2) with linoleic acidlevels of 10.2% and 8.4%, respectively. The fatty acid composition ofthis low linoleic acid mutant, which was stable to the M₇ generationunder both field and greenhouse conditions, is summarized in Table 13.In multiple location replicated yield trials, A133 was not significantlydifferent in yield from the parent cultivar Westar. An additional lowlinoleic acid line, designated M3062.8 (ATCC 75025), was also producedby the disclosed method. A 10-seed bulk analysis of this line showed thefollowing fatty acid composition: C_(16:0)—3.8%, C_(18:0)—2.3%,C_(18:1)—77.1%, C_(18:2)—8.9%, C_(18:3)—4.3%, FDA Sats—6.1%. This linehas also stably maintained its mutant fatty acid composition in thefield and greenhouse. TABLE 13 Fatty Acid Composition of a Low LinoleicAcid Canola Line Produced by Seed Mutagenesis Percent Fatty AcidsGenotype C_(16:0) C_(18:0) C_(18:1) C_(18:2) C_(18:3) Sats^(b) Westar3.9 1.9 67.5 17.6 7.4 7.0 W12638.8 3.9 2.3 75.0 9.4 6.1 7.5 (M₃)W12638.8.1 4.1 1.7 74.6 10.2 5.9 7.1 (M₄) A133.1.8 3.8 2.0 77.7 8.4 5.07.0 (M₅)^(a)Letter and numbers up to second decimal point indicate the plantline. Number after second decimal point indicates an individual plant.^(b)Sats = Total Saturate Content

EXAMPLE 4 Low Linolenic and Linoleic Acid Canola

In the studies of Example 1, at the M₃ generation, 57 lines exceeded thelower statistical threshold for linolenic acid (≦5.3%). Line W14749.8had 5.3% linolenic acid and 15.0% linoleic acid. At the M₄ and M₅generations, its selfed progenies [W14749.8, since designated M3032(ATCC 75021)] continued to exceed the statistical threshold for lowC_(18:3) with linolenic acid levels of 2.7% and 2.3%, respectively, andfor a low sum of linolenic and linoleic acids with totals of 11.8% and12.5% respectively. The fatty acid composition of this low linolenicacid plus linoleic acid mutant, which was stable to the M₅ generationunder both field and greenhouse conditions, is summarized in Table 14.In a single location replicated yield trial M3032 was not significantlydifferent in yield from the parent cultivar (Westar). TABLE 14 FattyAcid Composition of a Low Linolenic Acid Canola Line Produced by SeedMutagenesis Percent Fatty Acids Genotype C_(16:0) C_(18:0) C_(18:1)C_(18:2) C_(18:3) Sats Westar 3.9 1.9 67.5 17.6 7.4 7.0 W14749.8 4.0 2.569.4 15.0 5.3 6.5 (M₃) M3032.8 3.9 2.4 77.9 9.1 2.7 6.4 (M₄) M3032.1 3.52.8 80.0 10.2 2.3 6.5 (M₅)Sats = Total Saturate Content

EXAMPLE 5 Canola Lines Q508 and Q4275

Seeds of the B. napus line IMC-129 were mutagenized with methylN-nitrosoguanidine (MNNG). The MNNG treatment consisted of three parts:pre-soak, mutagen application, and wash. A 0.05M Sorenson's phosphatebuffer was used to maintain pre-soak and mutagen treatment pH at 6.1.Two hundred seeds were treated at one time on filter paper (Whatman #3M)in a petri dish (100 mm x 15 mm). The seeds were pre-soaked in 15 mls of0.05M Sorenson's buffer, pH 6.1, under continued agitation for twohours. At the end of the pre-soak period, the buffer was removed fromthe plate.

A 10 mM concentration of MNNG in 0.05M Sorenson's buffer, pH 6.1, wasprepared prior to use. Fifteen ml of 10 m MNNG was added to the seeds ineach plate. The seeds were incubated at 22° C.±3° C. in the dark underconstant agitation for four (4) hours. At the end of the incubationperiod, the mutagen solution was removed.

The seeds were washed with three changes of distilled water at 10 minuteintervals. The fourth wash was for thirty minutes. This treatment regimeproduced an LD60 population.

Treated seeds were planted in standard greenhouse potting soil andplaced into an environmentally controlled greenhouse. The plants weregrown under sixteen hours of light. At flowering, the racemes werebagged to produce selfed seed. At maturity, the M2 seed was harvested.Each M2 line was given an identifying number. The entire MNNG-treatedseed population was designated as the Q series.

Harvested M2 seeds was planted in the greenhouse. The growth conditionswere maintained as previously described. The racemes were bagged atflowering for selfing. At maturity, the selfed M3 seed was harvested andanalyzed for fatty acid composition. For each M3 seed line,approximately 10-15 seeds were analyzed in bulk as described in Example1.

High oleic-low linoleic M3 lines were selected from the M3 populationusing a cutoff of >82% oleic acid and <5.0% linoleic. From the first1600 M3 lines screened for fatty acid composition, Q508 was identified.The Q508 M3 generation was advanced to the M4 generation in thegreenhouse. Table 15 shows the fatty acid composition of Q508 and IMC129. The M4 selfed seed maintained the selected high oleic-low linoleicacid phenotype (Table 16). TABLE 15 Fatty Acid Composition of A129 andHigh Oleic Acid M3 Mutant Q508 Line # 16:0 18:0 18:1 18:2 18:3 A129* 4.02.4 77.7 7.8 4.2 Q508 3.9 2.1 84.9 2.4 2.9*Fatty acid composition of A129 is the average of 50 self-pollinatedplants grown with the M3 population

M₄ generation Q508 plants had poor agronomic qualities in the fieldcompared to Westar. Typical plants were slow growing relative to Westar,lacked early vegetative vigor, were short in stature, tended to bechlorotic and had short pods. The yield of Q508 was very low compared toWestar.

The M₄ generation Q508 plants in the greenhouse tended to be reduced invigor compared to Westar. However, Q508 yields in the greenhouse weregreater than Q508 yields in the field. TABLE 16 Fatty Acid Compositionof Seed Oil from Greenhouse-Grown Q508, IMC 129 and Westar. FDA Line16:0 18:0 18:1 18:2 18:3 Sats IMC 4.0 2.4 77.7 7.8 4.2 6.4 129^(a)Westar^(b) 3.9 1.9 67.5 17.6 7.4 >5.8 Q508^(c) 3.9 2.1 84.9 2.4 2.9 6.0^(a)Average of 50 self-pollinated plants^(b)Data from Example 1^(c)Average of 50 self-pollinated plants

Nine other M4 high-oleic low-linoleic lines were also identified: Q3603,Q3733, Q4249, Q6284, Q6601, Q6761, Q7415, Q4275, and Q6676. Some ofthese lines had good agronomic characteristics and an elevated oleicacid level in seeds of about 80% to about 84%.

Q4275 was crossed to the variety Cyclone. After selfing for sevengenerations, mature seed was harvested from 93GS34-179, a progeny lineof the Q4275 Cyclone cross. Referring to Table 17, fatty acidcomposition of a bulk seed sample shows that 93GS34 retained the seedfatty acid composition of Q4275. 93GS34-179 also maintainedagronomically desirable characteristics.

After more than seven generations of selfing of Q4275, plants of Q4275,IMC 129 and 93GS34 were field grown during the summer season. Theselections were tested in 4 replicated plots (5 feet×20 feet) in arandomized block design. Plants were open pollinated. No-selfed seed wasproduced. Each plot was harvested at maturity, and a sample of the bulkharvested seed from each line was analyzed for fatty acid composition asdescribed above. The fatty acid compositions of the selected lines areshown in Table 17. TABLE 17 Fatty Acid Composition of Field Grown IMC129, Q4275 and 93GS34 Seeds Fatty Acid Composition (%) Line C_(16:0)C_(18:0) C_(18:1) C_(18:2) C_(18:3) FDA Sats IMC 129 3.3 2.4 76.7 8.75.2 5.7 Q4275 3.7 3.1 82.1 4.0 3.5 6.8 93GS34-179 2.6 2.7 85.0 2.8 3.35.3

The results shown in Table 17 show that Q4275 maintained the selectedhigh oleic—low linoleic acid phenotype under field conditions. Theagronomic characteristics of Q4275 plants were superior to those ofQ508.

M₄ generation Q508 plants were crossed to a dihaploid selection ofWestar, with Westar serving as the female parent. The resulting F1 seedwas termed the 92EF population. About 126 F1 individuals that appearedto have better agronomic characteristics than the Q508 parent wereselected for selfing. A portion of the F₂ seed from such individuals wasreplanted in the field. Each F2 plant was selfed and a portion of theresulting F3 seed was analyzed for fatty acid composition. The contentof oleic acid in F₃ seed ranged from 59 to 79%. No high oleic (>80%)individuals were recovered with good agronomic type.

A portion of the F₂ seed of the 92EF population was planted in thegreenhouse to analyze the genetics of the Q508 line. F₃ seed wasanalyzed from 380 F2 individuals. The C_(18:1) levels of F₃ seed fromthe greenhouse experiment is depicted in FIG. 1. The data were testedagainst the hypothesis that Q508 contains two mutant genes that aresemi-dominant and additive: the original IMC 129 mutation as well as oneadditional mutation. The hypothesis also assumes that homozygous Q508has greater than 85% oleic acid and homozygous Westar has 62-67% oleicacid. The possible genotypes at each gene in a cross of Q508 by Westarmay be designated as:

-   -   AA=Westar Fad2^(a)    -   BB=Westar Fad2^(b)    -   aa=Q508 Fad2^(a−)    -   bb=Q508 Fad2^(b−)

Assuming independent segregation, a 1:4:6:4:1 ratio of phenotypes isexpected. The phenotypes of heterozygous plants are assumed to beindistinguishable and, thus, the data were tested for fit to a 1:14:1ratio of homozygous Westar: heterozygous plants: homozygous Q508.Phenotypic # of Ratio Westar Alleles Genotype 1 4 AABB(Westar) 4 3 AABb,AaBB, AABb, AaBB 6 2 AaBb, AAbb, AaBb, AaBb, aaBB, AaBb 4 1 Aabb, aaBb,Aabb, aaBb 1 0 aabb (Q508)

Using Chi-square analysis, the oleic acid data fit a 1:14:1 ratio. Itwas concluded that Q508 differs from Westar by two major genes that aresemi-dominant and additive and that segregate independently. Bycomparison, the genotype of IMC 129 is aaBB.

The fatty acid composition of representative F3 individuals havinggreater than 85% oleic acid in seed oil is shown in Table 18. The levelsof saturated fatty acids are seen to be decreased in such plants,compared to Westar. TABLE 18 92EF F₃ Individuals with >85% C_(18:1) inSeed Oil F3 Plant Fatty Acid Composition (%) Identifier C16:0 C18:0C18:1 C18:2 C18:3 FDASA +38068 3.401 1.582 85.452 2.134 3.615 4.983+38156 3.388 1.379 85.434 2.143 3.701 4.767 +38171 3.588 1.511 85.2892.367 3.425 5.099 +38181 3.75 1.16 85.312 2.968 3.819 4.977 +38182 3.5290.985 85.905 2.614 3.926 4.56 +38191 3.364 1.039 85.737 2.869 4.0394.459 +38196 3.557 1.182 85.054 2.962 4.252 4.739 +38202 3.554 1.10586.091 2.651 3.721 4.713 +38220 3.093 1.16 86.421 1.931 3.514 4.314+38236 3.308 1.349 85.425 2.37 3.605 4.718 +38408 3.617 1.607 85.34 2.333.562 5.224 +38427 3.494 1.454 85.924 2.206 3.289 4.948 +38533 3.641.319 85.962 2.715 3.516 4.959

EXAMPLE 6 Leaf and Root Fatty Acid Profiles of Canola Lines IMC-129,Q508, and Westar

Plants of Q508, IMC 129 and Westar were grown in the greenhouse. Matureleaves, primary expanding leaves, petioles and roots were harvested atthe 6-8 leaf stage, frozen in liquid nitrogen and stored at −70° C.Lipid extracts were analyzed by GLC as described in Example 1. The fattyacid profile data are shown in Table 19.

The data in Table 19 indicate that total leaf lipids in Q508 are higherin C_(18:1) content than the C_(18:2) plus C_(18:3) content. The reverseis true for Westar and IMC 129. The difference in total leaf lipidsbetween Q508 and IMC 129 is consistent with the hypothesis that a secondFad2 gene is mutated in Q508.

The C_(16:3) content in the total lipid fraction was about the same forall three lines, suggesting that the plastid FadC gene product was notaffected by the Q508 mutations. To confirm that the FadC gene was notmutated, chloroplast lipids were separated and analyzed. No changes inchloroplast C_(16:1), C_(16:2) or C_(16:3) fatty acids were detected inthe three lines. The similarity in plastid leaf lipids among Q508,Westar and IMC 129 is consistent with the hypothesis that the secondmutation in Q508 affects a microsomal Fad2 gene and not a plastid FadCgene. TABLE 19 MATURE EXPANDING LEAF LEAF PETIOLE ROOT West. 129 3Q508West. 129 3Q508 West. 129 3Q508 West. 129 3Q508 16:0 12.1 11.9 10.1 16.416.1 11.3 21.7 23.5 11.9 21.1 21.9 12.0 16:1 0.8 0.6 1.1 0.7 0.6 1.1 1.01.3 1.4 — — — 16:2 2.3 2.2 2.0 2.8 3.1 2.8 1.8 2.2 1.8 — — — 16:3 14.715.0 14.0 6.3 5.4 6.9 5.7 4.6 5.7 — — — 18:0 2.2 1.6 1.2 2.5 2.8 1.5 3.74.0 1.6 3.6 2.9 2.5 18:1 2.8 4.9 16.7 3.8 8.3 38.0 4.9 12.9 46.9 3.5 6.168.8 18:2 12.6 11.5 6.8 13.3 13.8 4.9 20.7 18.3 5.2 28.0 30.4 4.4 18:350.6 50.3 46.0 54.2 50.0 33.5 40.4 33.2 25.3 43.8 38.7 12.3

EXAMPLE 7 Sequences of Mutant and Wild-Type Delta-12 Fatty AcidDesaturases from B. napus

Primers specific for the FAD2 structural gene were used to clone theentire open reading frame (ORF) of the D and F delta-12 desaturase genesby reverse transcriptase polymerase chain reaction (RT-PCR). RNA fromseeds of IMC 129, Q508 and Westar plants was isolated by standardmethods and was used as template. The RT-amplified fragments were usedfor nucleotide sequence determination. The DNA sequence of each genefrom each line was determined from both strands by standard dideoxysequencing methods.

Sequence analysis revealed a G to A transversion at nucleotide 316 (fromthe translation initiation codon) of the D gene in both IMC 129 andQ508, compared to the sequence of Westar. The transversion changes thecodon at this position from GAG to AAG and results in a non-conservativesubstitution of glutamic acid, an acidic residue, for lysine a basicresidue. The presence of the same mutation in both lines was expectedsince the Q508 line was derived from IMC 129. The same base change wasalso detected in Q508 and IMC 129 when RNA from leaf tissue was used astemplate.

The G to A mutation at nucleotide 316 was confirmed by sequencingseveral independent clones containing fragments amplified directly fromgenomic DNA of IMC 129 and Westar. These results eliminated thepossibility of a rare mutation introduced during reverse transcriptionand PCR in the RT-PCR protocol. It was concluded that the IMC 129 mutantis due to a single base transversion at nucleotide 316 in the codingregion of the D gene of rapeseed microsomal delta 12-desaturase.

A single base transition from T to A at nucleotide 515 of the F gene wasdetected in Q508 compared to the Westar sequence. The mutation changesthe codon at this position from CTC to CAC, resulting in thenon-conservative substitution of a non-polar residue, leucine, for apolar residue, histidine, in the resulting gene product. No mutationswere found in the F gene sequence of IMC 129 compared to the F genesequence of Westar.

These data support the conclusion that a mutation in a delta-12desaturase gene sequence results in alterations in the fatty acidprofile of plants containing such a mutated gene. Moreover, the datashow that when a plant line or species contains two delta-12 desaturaseloci, the fatty acid profile of an individual having two mutated locidiffers from the fatty acid profile of an individual having one mutatedlocus.

The mutation in the D gene of IMC 129 and Q508 mapped to a region havinga conserved amino acid motif (His-Xaa-Xaa-Xaa-His) found in cloneddelta-12 and delta-15 membrane bound-desaturases (Table 20). TABLE 20Alignment of Amino Acid Sequences of Cloned Canola MembraneBound-Desaturases Desaturase Gene Sequence^(a) PositionCanola-fad2-D(mutant) AHKCGH 109-114 Canola-Fad2-D AHECGH 109-114Canola-Fad2-F AHECGH 109-114 Canola-FadC GHDCAH 170-175 Canola-fad3(mutant) GHKCGH 94-99 Canola-Fad3 GHDCGH 94-99 Canola-FadD GHDCGH125-130(FadD = Plastid delta 15, Fad3 = Microsomal delta-15),(FadC = Plastid delta-12, Fad2 = Microsomal delta-12)^(a)One letter amino acid code; conservative substitutions areunderlined; non-conservative substitutions are in bold.

EXAMPLE 8 Transcription and Translation of Microsomal Delta-12 FattyAcid Desaturases

Transcription in vivo was analyzed by RT-PCR analysis of stage II andstage III developing seeds and leaf tissue. The primers used tospecifically amplify delta-12 desaturase F gene RNA from the indicatedtissues were sense primer 5′-GGATATGATGATGGTGAAAGA-3′ and antisenseprimer 5′-TCTTTCACCATCATCATATCC-3′. The primers used to specificallyamplify delta-12 desaturase D gene RNA from the indicated tissues weresense primer 5′-GTTATGAAGCAAAGAAGAAAC-3′ and antisense primer5′-GTTTCTTCTTTGCTTCATAAC-3′. The results indicated that mRNA of both theD and F gene was expressed in seed and leaf tissues of IMC 129, Q508 andwild type Westar plants.

In vitro transcription and translation analysis showed that a peptide ofabout 46 kD was made. This is the expected size of both the D geneproduct and the F gene product, based on sum of the deduced amino acidsequence of each gene and the cotranslational addition of a microsomalmembrane peptide.

These results rule out the possibility that non-sense or frameshiftmutations, resulting in a truncated polypeptide gene product, arepresent in either the mutant D gene or the mutant F gene. The data, inconjunction with the data of Example 7, support the conclusion that themutations in Q508 and IMC 129 are in delta-12 fatty acid desaturasestructural genes encoding desaturase enzymes, rather than in regulatorygenes.

EXAMPLE 9 Development of Gene-Specific PCR Markers

Based on the single base change in the mutant D gene of IMC 129described in above, two 5′ PCR primers were designed. The nucleotidesequence of the primers differed only in the base (G for Westar and Afor IMC 129) at the 3′ end. The primers allow one to distinguish betweenmutant fad2-D and wild-type Fad2-D alleles in a DNA-based PCR assay.Since there is only a single base difference in the 5′ PCR primers, thePCR assay is very sensitive to the PCR conditions such as annealingtemperature, cycle number, amount, and purity of DNA templates used.Assay conditions have been established that distinguish between themutant gene and the wild type gene using genomic DNA from IMC 129 andwild type plants as templates. Conditions may be further optimized byvarying PCR parameters, particularly with variable crude DNA samples. APCR assay distinguishing the single base mutation in IMC 129 from thewild type gene along with fatty acid composition analysis provides ameans to simplify segregation and selection analysis of genetic crossesinvolving plants having a delta-12 fatty acid desaturase mutation.

EXAMPLE 10 Transformation with Mutant and Wild Type Fad3 Genes

B. napus cultivar Westar was transformed with mutant and wild type Fad3genes to demonstrate that the mutant Fad3 gene for canola cytoplasmiclinoleic desaturase delta-15 desaturase is nonfunctional. Transformationand regeneration were performed using disarmed Agrobacterium tumefaciensessentially following the procedure described in WO 94/11516.

Two disarmed Agrobacterium strains were engineered, each containing a Tiplasmid having the appropriate gene linked to a seed-specific promoterand a corresponding termination sequence. The first plasmid, pIMC110,was prepared by inserting into a disarmed Ti vector the full length wildtype Fad3 gene in sense orientation (nucleotides 208 to 1336 of SEQ ID 6in WO 93/11245), flanked by a napin promoter sequence positioned 5′ tothe Fad3 gene and a napin termination sequence positioned 3′ to the Fad3gene. The rapeseed napin promoter is described in EP 0255378.

The second plasmid, pIMC205, was prepared by inserting a mutated Fad3gene in sense orientation into a disarmed Ti vector. The mutant sequencecontained mutations at nucleotides 411 and 413 of the microsomal Fad3gene described in W093/11245, thus changing the sequence for codon 96from GAC to AAG. The amino acid at codon 96 of the gene product wasthereby changed from aspartic acid to lysine. See Table 20. A bean(Phaseolus vulgaris) phaseolin (7S seed storage protein) promoterfragment of 495 base pairs, starting with 5′-TGGTCTTTTGGT-3′, was placed5′ to the mutant Pad3 gene and a phaseolin termination sequence wasplaced 3′ to the mutant Fad3 gene. The phaseolin sequence is describedin Doyle et al., (1986) J. Biol. Chem. 261:9228-9238) and Slightom etal., (1983) Proc. Natl. Acad. Sci. USA 80:1897-1901.

The appropriate plasmids were engineered and transferred separately toAgrobacterium strain LBA4404. Each engineered strain was used to infect5 mm segments of hypocotyl explants from Westar seeds by cocultivation.Infected hypocotyls were transferred to callus medium and, subsequently,to regeneration medium. Once discernable stems formed from the callus,shoots were excised and transferred to elongation medium. The elongatedshoots were cut, dipped in Rootone™, rooted on an agar medium andtransplanted to potting soil to obtain fertile T1 plants. T2 seeds wereobtained by selfing the resulting T1 plants.

Fatty acid analysis of T2 seeds was carried out as described above. Theresults are summarized in Table 21. Of the 40 transformants obtainedusing the pIMC110 plasmid, 17 plants demonstrated wild type fatty acidprofiles and 16 demonstrated overexpression. A proportion of thetransformants are expected to display an overexpression phenotype when afunctioning gene is transformed in sense orientation into plants.

Of the 307 transformed plants having the pIMC205 gene, none exhibited afatty acid composition indicative of overexpression. This resultindicates that the mutant fad3 gene product is non-functional, sincesome of the transformants would have exhibited an overexpressionphenotype if the gene product were functional. TABLE 21 Overexpressionand Co-suppression Events in Westar Populations Transformed with pIMC205or pIMC110. α-Linolenic Overexpression Co-Suppression Number of AcidEvents Events Wild Type Construct Transformants Range (%) (>10%linolenic) (<4.0% linolenic) Events pIMC110 40 2.4-20.6 16 7 17 pIMC205307 4.6-10.4 0 0 307

Fatty acid compositions of representative transformed plants arepresented in Table 22. Lines 652-09 and 663-40 are representative ofplants containing pIMC110 and exhibiting an overexpression and aco-suppression phenotype, respectively. Line 205-284 is representativeof plants containing pIMC205 and having the mutant fad3 gene. TABLE 22Fatty Acid Composition of T2 Seed From Westar Transformed With pIMC205or pIMC110. Fatty Acid Composition (%) Line C16:0 C18:0 C18:1 C18:2C18:3 652-09 pIMC110 4.7 3.3 65.6 8.1 14.8 overexpression 663-40 4.9 2.162.5 23.2 3.6 PIMC110 co-suppression 205-284 3.7 1.8 68.8 15.9 6.7pIMC205

EXAMPLE 11 Sequences of Wild Type and Mutant Fad2-D and Fad2-F

High molecular weight genomic DNA was isolated from leaves of Q4275plants (Example 5). This DNA was used as template for amplification ofFad2-D and Fad2-F genes by polymerase chain reaction (PCR). PCRamplifications were carried out in a total volume of 100 μl andcontained 0.3 μg genomic DNA, 200 μM deoxyribonucleoside triphosphates,3 mM MgSO₄, 1-2 Units DNA polymerase and 1×Buffer (supplied by the DNApolymerase manufacturer). Cycle conditions were: 1 cycle for 1 min at95° C., followed by 30 cycles of 1 min at 94° C., 2 min at 55° C. and 3min at 73° C.

The Fad2-D gene was amplified once using Elongase® (Gibco-BRL). PCRprimers were:

-   CAUCAUCAUCAUCTTCTTCGTAGGGTTCATCG and-   CUACUACUACUATCATAGAAGAGAAAGGTTCAG for the 5′ and 3′ ends of the    gene, respectively.

The Fad2-F gene was independently amplified 4 times, twice withElongase® and twice with Taq polymerase (Boehringer Mannheim). The PCRprimers used were:

-   5′CAUCAUCAUCAUCATGGGTGCACGTGGAAGAA3′ and-   5′CUACUACUACUATCTTTCACCATCATCATATCC3′ for the 5′ and 3′ ends of the    gene, respectively.

Amplified DNA products were resolved on an agarose gel, purified byJetSorb® and then annealed into pAMP1 (Gibco-BRL) via the (CAU)₄ and(CUA)₄ sequences at the ends of the primers, and transformed into E.coli DH5α.

The Fad2-D and Fad2-F inserts were sequenced on both strands with an ABIPRISM 310 automated sequencer (Perkin-Elmer) following themanufacturer's directions, using synthetic primers, AmpliTaq® DNApolymerase and dye terminator.

The Fad2-D gene was found to have an intron upstream of the ATG startcodon. As expected, the coding sequence of the gene contained a G to Amutation at nucleotide 316, derived from IMC 129 (FIG. 2).

A single base transversion from G to A at nucleotide 908 was detected inthe F gene sequence of the Q4275 amplified products, compared to thewild type F gene sequence (FIG. 2). This mutation changes the codon atamino acid 303 from GGA to GAA, resulting in the non-conservativesubstitution of a glutamic acid residue for a glycine residue (Table 3and FIG. 3). Expression of the mutant Q4275 Fad2-F delta-12 desaturasegene in plants alters the fatty acid composition, as describedhereinabove.

To the extent not already indicated, it will be understood by those ofordinary skill in the art that any one of the various specificembodiments herein described and illustrated may be further modified toincorporate features shown in other of the specific embodiments.

The foregoing detailed description has been provided for a betterunderstanding of the invention only and no unnecessary limitation shouldbe understood therefrom as some modifications will be apparent to thoseskilled in the art without deviating from the spirit and scope of theappended claims.

1-34. (canceled)
 35. An isolated nucleic acid fragment comprising asequence of at least about 20 nucleotides from a Brassicaceae delta-15fatty acid desaturase gene having at least one mutation, wherein said atleast one mutation is effective for increasing levels of oleic acid inBrassicaceae seeds and wherein said sequence includes said at least onemutation.
 36. The nucleic acid fragment of claim 35, wherein saidsequence comprises a full-length coding sequence of said gene.
 37. Thenucleic acid fragment of claim 35, wherein said mutant desaturase geneencodes a microsomal gene product.
 38. The nucleic acid fragment ofclaim 35, wherein said at least one mutation comprises a mutation in aregion of said desaturase gene encoding a His-Xaa-Xaa-Xaa-His amino acidmotif.
 39. The nucleic acid fragment of claim 38, wherein said at leastone mutation comprises a non-conservative amino acid substitution insaid region.
 40. The nucleic acid fragment of claim 39, wherein saidmotif comprises the sequence His-Asp-Cys-Gly-His.
 41. The nucleic acidfragment of claim 40, wherein said at least one mutation comprises thesequence His-Lys-Cys-Gly-His.
 42. The nucleic acid fragment of claim 35,wherein said mutant desaturase gene is from a Brassica napus plant. 43.A Brassicaceae plant containing a sequence of at least 20 nucleotidesfrom a delta-15 fatty acid desaturase gene having at least one mutation,said at least one mutation in a region encoding a His-Xaa-Xaa-Xaa-Hisamino acid motif and wherein said mutation confers an altered fatty acidcomposition in seeds of said plant.
 44. The plant of claim 43, whereinsaid plant contains a full-length coding sequence of said mutant gene.45. The plant of claim 43, wherein said mutation confers a decreasedlevel of α-linolenic acid in said seeds.
 46. The plant of claim 43,wherein said mutant desaturase gene encodes a microsomal gene product.47. The plant of claim 43, wherein said at least one mutation comprisesa non-conservative amino acid substitution in said region.
 48. The plantof claim 47, wherein said motif comprises the sequenceHis-Asp-Cys-Gly-His.
 49. The plant of claim 48, wherein said at leastone mutation comprises the sequence His-Lys-Cys-Gly-His.
 50. The plantof claim 43, wherein said mutant desaturase gene is from a Brassicanapus plant.
 51. The plant of claim 43, wherein said plant is a Brassicanapus plant.
 52. A Brassicaceae plant containing: a) a sequence of atleast 20 nucleotides from a delta-15 fatty acid desaturase gene havingat least one mutation, said at least one delta-15 gene mutation in aregion encoding a His-Xaa-Xaa-Xaa-His amino acid motif; b) a sequence ofat least 20 nucleotides from a delta-12 fatty acid desaturase genehaving at least one mutation, said at least one delta-12 gene mutationin a region encoding a His-Xaa-Xaa-Xaa-His amino acid motif; and c) saiddelta-15 gene mutation and said delta-12 gene mutation conferring analtered fatty acid composition in seeds of said plant.
 53. The plant ofclaim 52, wherein said mutant genes confer a decreased level ofα-linolenic acid in said seeds.
 54. A vegetable oil extracted from seedsproduced by the plant of claim
 43. 55. The oil of claim 54, wherein,following crushing of said seeds and extraction of said oil, said seedssaid oil has from about 0.5% to about 10.0% α-linolenic acid based ontotal fatty acid composition.
 56. A vegetable oil extracted from seedsproduced by the plant of claim
 52. 57. A vegetable oil extracted fromseeds produced by the plant of claim
 53. 58. A method for producing aBrassicaceae plant line, comprising the steps of: a) inducingmutagenesis in cells of a starting variety of a Brassicaceae species; b)obtaining progeny plants from said cells; c) identifying at least one ofsaid progeny plants that contains a delta-15 fatty acid desaturase genehaving at least one mutation, said at least one mutation in a regionencoding a His-Xaa-Xaa-Xaa-His amino acid motif; d) producing said plantline from said at least one progeny plant by self-pollination for atleast three additional generations.
 59. The method of claim 58, whereinsaid identifying step comprises a technique selected from the groupconsisting of: PCR, 3SR, and direct polynucleotide sequencing.